Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

1. Preparing the Solutions and Reagents

1. Amino acid stock solution
   1. Prepare a 4X amino acid stock solution^23,24 by weighing and adding the 20 different amino acids to an Erlenmeyer flask according to the concentrations given in Table 1.
      NOTE: In some cases, amino acid(s) can be excluded from the solution and replaced by equal molar amounts of mannitol to maintain an equal concentration of osmolytes.
   2. Add the appropriate amount of ddH₂O to produce the desired volume of solution.
   3. To ensure that the amino acids have completely dissolved, heat gently while continually stirring until the liquid is completely clear; it will take on a slight yellow hue.
      NOTE: It may be necessary to reduce the pH to 6 by adding HCl to allow some of the more hydrophobic amino acids to dissolve.
   4. Aliquot the solution and sterile-filter using a syringe filter with a 0.2-µm pore size.
   5. Store in a sealed container at -20 °C for up to 6 months.
2. APS
   Note: See²⁵.
   1. For the 20X salt stock solution, combine the salts listed in Table 2 in 50 mL of water. Stir until completely dissolved.
      NOTE: It may be necessary to slightly heat the solution to completely dissolve the salts.
   2. Sterile-filter using a syringe filter with a 0.2-µm pore size and store the solution in a 50-mL tube at -20 °C.
3. Calcium stock solution
   1. For the 50X calcium stock solution, add 0.90 g of calcium chloride to 100 mL of water (Table 3); stir until completely dissolved.
   2. Sterile-filter using a syringe filter with a 0.2-µm pore size, aliquot the solution into 50-mL tubes, and store at -20 °C.
4. Tris buffer (pH 7.4)
   1. For the Tris buffer, combine the salt and solutions listed in Table 4 and add ddH₂O up to 100 mL.
   2. Sterile-filter using a syringe filter with a 0.2-µm pore size and aliquot the solution into 50-mL tubes; store at room temperature.
5. Fat body culture medium
   1. To prepare the fat body culture medium, prepare all solutions listed above.
   2. Combine them as per the volumes in Table 5 to make 200 mL of fat body culture medium.
   3. Adjust the pH to 7.2 using NaOH or HCl.
   4. Sterile-filter the solution using a syringe filter with a 0.2-µm pore size.
   5. Divide the solution into 15-mL aliquots and store at -20 °C for up to 6 months.

2. Preparing for Dissection

1. Prepare a stereomicroscope (10-20x magnification) with illumination and lay out two ultra-fine tweezers and a pair of micro dissection scissors.
2. Prepare all solutions necessary for the experiment. Thaw the fat body culture medium to room temperature.
   NOTE: Precipitants can be dissolved by gently heating the solution to no higher than 30 °C.
3. With an aspirator, collect adult female mosquitoes (3-7 days after emergence) and anesthetize them using carbon dioxide or ice.
   NOTE: Once anesthetized, the mosquitoes should be kept under CO₂ for the shortest time possible, not exceeding 20 min. Alternatively, the mosquitoes can be anesthetized on ice and kept for approximately 30 min.
4. Prepare a 6-well plate with 3 mL of APS per well.

3. Fat Body Dissection

1. Focus the dissecting stereomicroscope and adjust the chair to a comfortable height.
2. Place a concave microscope slide on the microscope surface and add two drops of APS to the center.
3. Pick up a mosquito by a leg using a forceps and transfer it to the APS surface on the microscope slide.
4. Carefully grip the mosquito by the thorax, with the forceps in the left hand, and rotate the mosquito so that the ventral side is upwards.
5. While holding the body steady by the thorax, grasp the last two abdominal segments, with the forceps in the right hand, and gently pull.
   NOTE: The last two abdominal segments will separate from the abdomen and the attached ovaries, Malpighian tubules, hindgut, and midgut; sometimes the crop will slide out of the abdomen. If they have not been removed, insert the closed tips of the forceps into the small hole created and remove the remaining tissues.
6. Set the right forceps aside and pick up the spring scissors. Slide one blade into the hole in the abdomen up to the segment below the thorax. Gently and swiftly cut the abdomen lengthwise.
   NOTE: Once the cut has been made, the abdomen should begin to expand outward, though this may not occur immediately.
7. Proceed to make the second cut, which will be a lateral cut below the thorax and slightly below where the first cut ended.
   NOTE: The abdomen should then open up and dissociate from the thorax, with the cuticle up and the fat bodies side-immersed within the APS. This abdominal body wall is the fat body preparation for in vitro culture.
8. Lift the fat bodies from the APS solution using the tip of a pair of forceps and transfer them from the solution to the 6-well plate containing APS at room temperature. Allow the tissue to rest for approximately 0.5 h before advancing to the fat body culture.

4. Fat Body Culture

1. Incubate the fat bodies on APS at room temperature for at least 0.5 h to equilibrate them.
2. Transfer the fat bodies using the tip of the forceps and gently lift them out of the APS solution. Lower the tip into the fat body culture medium.
   NOTE: The fat body should open on the surface of the medium and float freely. The fat body culture is typically performed in 96-well plates, with 150-200 µL of medium in each well. One well can fit up to three individual fat bodies, and they are viable for more than 12 h (unpublished personal results).
3. After the incubation period, transfer the fat bodies from the medium into 1.5-mL centrifuge tubes containing the appropriate reagents for downstream processing.
   NOTE: Typical analyses include qPCR^16,17,18,19, Western blotting^18,20, transcriptomics²⁶, proteomics²¹, or metabolomics²².